ABSTRACT
Molecular mechanisms determining methylation patterns in eukaryotic genomes still remain unresolved. We have characterized, in Ascobolus, a gene for de novo methylation. This novel eukaryotic gene, masc1, encodes a protein that has all motifs of the catalytic domain of eukaryotic C5-DNA-methyltransferases but is unique in that it lacks a regulatory N-terminal domain. The disruption of masc1 has no effect on viability or methylation maintenance but prevents the de novo methylation of DNA repeats, which takes place after fertilization, through the methylation induced premeiotically (MIP) process. Crosses between parents harboring the masc1 disruption are arrested at an early stage of sexual reproduction, indicating that the activity of Masc1, the product of the gene, is crucial in this developmental process.
Subject(s)
Ascomycota/genetics , DNA (Cytosine-5-)-Methyltransferases/genetics , DNA Methylation , DNA-Binding Proteins , Fungal Proteins , Methyltransferases/genetics , Amino Acid Sequence , Animals , Arabidopsis , Base Sequence , Cloning, Molecular , DNA (Cytosine-5-)-Methyltransferases/chemistry , DNA (Cytosine-5-)-Methyltransferases/metabolism , Eukaryotic Cells/enzymology , Gene Expression Regulation, Enzymologic/genetics , Genetic Complementation Test , Homozygote , Mice , Molecular Sequence Data , Mutation/physiology , Reproduction, Asexual/physiology , Sequence Homology, Amino AcidABSTRACT
The filamentous fungus Ascobolus immersus represents an eukaryotic model organism to study genetic phenomena linked to DNA methylation. Following our previous characterization of a gene, masc1 from A. immersus, encoding the 'de novo' C5-DNA-methyltransferase (MTase), we report here the identification of a second MTase gene, masc2. The deduced peptide sequence of Masc2 is similar to previously identified eukaryotic MTases and distinct from Masc1 by having a large N-terminal domain in addition to the ubiquitous C-terminal catalytic domain. Following cloning of the gene, Masc2 was overexpressed and purified. Masc2 shows MTase activity with double stranded DNAs. Structural and biochemical properties of Masc2 suggest that it may function as a 'maintenance' MTase. With this finding, A. immersus represents so far the only eukaryotic organism in which two possibly synergistically operating MTases have been identified.
